This application is a 371 of PCT/JP00/03055 filed May 12, 2000, now WO 00/69868 published Nov. 23, 2000.
The present invention relates to physiologically active substances, sulphostin and a sulphostin analogue and a process for producing the same. The compounds in accordance with the present invention possess a dipeptidylpeptidase IV inhibiting activity and are expected to be a physiologically active substance to be used, for example, as immunomodulating agents, hormone-modulating agents, anti-HIV drugs, antiallergic drugs, anti-inflammatory drugs and antirheumatic drugs.
Dipeptidylpeptidase IV present on the surface of T cells is known to concern itself with the activation of T cells (Immunol. Today, 15, 180-184(1994)) and is playing an important role in an immune system. In addition, the dipeptidylpeptidase IV concerns itself with the decomposition of growth-hormone-releasing hormone (J. Clin. Invest., 83, 1533-1540(1989)).
Previously, diprotin A and B and the like are known as the physiologically active substances possessing a dipeptidylpeptidase IV inhibiting activity (J. Antibiotics, 37, 422-425(1984)).
However, it cannot be said that the inhibiting activity of diprotin A and B against enzymes is sufficiently high. Therefore, a physiologically active substance possessing a higher inhibiting activity has been desired.
The present inventors previously found sulphostin as such a physiologically active substance, and developed a process for producing the same in a manner such that microorganisms which belong to Sttreptomyces and are capable of producing sulphostin of a physiologically active substance, are cultured in a medium, and the physiologically active substance, sulphostin, produced and accumulated in the cultivated substance is collected (Japanese Patent Application No. 9-317221). However, the sulphostin can exhibit a strong inhibiting activity against enzymes, yet the process of collecting it from the cultivated substance of microorganisms is not always said to be high in productivity, and the process is not suitable for mass production.
The present inventors have satisfactorily clarified a chemical structure of the physiologically active substance, sulphostin, and found that it can be obtained according to a synthetic chemical means. Further, it has been found that stereo isomers and analogues of the sulphostin, which are not naturally occurring, can be obtained similarly according to a synthetic chemical means, and can exhibit a strong enzyme inhibiting activity like the sulphostin. The present invention has been accomplished on the basis of the above-mentioned knowledge.
The present invention provides a sulphostin analogue represented by a general formula (IVxe2x80x2) or a pharmaceutically acceptable salt thereof, 
wherein n is an integer of from 0 to 3, provided that a case where n is 2 and steric configurations of C* and P* are S and R, respectively, is excluded.
Further, the present invention provides a process for producing sulphostin or a sulphostin analogue represented by a general formula (IV), 
wherein n is an integer of from 0 to 3, which comprises allowing a compound represented by a general formula (II), 
wherein n is as defined above, and Z is an amino group-protecting group, to reaction with sulfur trioxide or a complex thereof, if necessary followed by cation exchange, thereby obtaining a compound represented by a general formula (III), 
wherein n and Z are as defined above, and M is a monovalent cation, and then removing the protecting group.
Still further, the present invention provides a process for producing a compound represented by a general formula (II), 
wherein n is an integer of from 0 to 3, and Z is an amino group-protecting group, which comprises allowing a compound represented by a general formula (I), 
wherein n and Z are as defined above, to react with a base, followed by reaction with phosphorus oxychloride or POX3 in which X is a halogen or imidazole, and further followed by reaction with ammonia in order.
The present inventors have clarified that according to a variety of spectrography, the sulphostin has a chemical structure represented by the following formula (V). In addition, on the basis of a fact that L-ornithine is obtained through an experiment comprising a hydrolysis thereof, it has been also clarified that a steric configuration at a joint of the amino group is S. 
According to such a knowledge, the sulphostin has been synthesized in a manner mentioned below.
L-Ornithine is esterified, followed by ring-closure, thereby obtaining L-ornithine lactam, whose amino group is then protected in a conventional manner to obtain a compound of a general formula (VI). 
In the formula, Z is a conventional amino group-protecting group, for example, carbamate type protecting groups such as a benzyloxycarbonyl group, whose benzyl group may be substituted with those such as a lower alkyl group, a lower alkoxy group, an acyloxy group, a nitro group and a halogen, and t-butoxycarbonyl group, amide type protecting groups such as formyl, acetyl and trifluoroacetyl, and imide type protecting groups such as phthaloyl. Preferred are carbamate type protecting groups, and more preferred is a benzyloxycarbonyl group.
Successively, the compound of the formula (VI) is treated in a non-aqueous solvent with a base, followed by reaction with POX3 in which X is an eliminating group such as a halogen and imidazole, preferably a halogen, and further followed by reaction with ammonia, thereby obtaining a compound of a general formula (VII). 
In the formula, Z is the same amino group-protecting group as in the general formula (VI). The solvent includes an aprotic solvent, and may be anything capable of dissolving the compound of the formula (VI). Preferred is an ether solvent such as THF. The base may be anything capable of substituting hydrogen of the lactam with a metal, and includes butyllithium, sodium hydride, lithium hydride, potassium hydride, sodium bistrimethylsilylamide and lithium bistrimethylsilylamide. Preferred is butyllithium. The reaction can be carried out at a temperature of from xe2x88x9280 to 100xc2x0 C., preferably from xe2x88x9280 to 0xc2x0 C.
Successively, the compound of the formula (VII) is allowed to react with sulfur trioxide or a complex thereof such as a pyridine complex, a DMF complex and a trimethylamine complex, at a temperature of from 0 to 100xc2x0 C., preferably from 0 to 10xc2x0 C., if desired followed by cation exchange, thereby obtaining a compound of a general formula (VIII). 
In the formula, Z is the same amino group-protecting group as in the general formula (VI), and M+ is a monovalent cation such as Li+, Na+, K+, R3NH+, R2NH2+, RNH3+ and pyridinium, in which R is a lower alkyl group substituted or unsubstituted with an aryl group such as phenyl and naphthyl. The solvent includes an aprotic solvent such as 1,2-dichloroethane, chloroform, methylene chloride, tetrahydrofuran, dioxane, DMF, dimethylacetamide, hexamethylphosphoramide (HMPA), N-methylpyrrolidone and acetonitrile. Preferred is dimethylformamide (DMF). The compound of the general formula (VIII) is a mixture of two diastereoisomers in relation to the asymmetric carbon at a joint of the protected amino group, because asymmetry is newly generated at the phosphorus atom. These isomers can be separated from each other in a manner such that an aqueous solution of sodium hydroxide, sodium carbonate, sodium hydrogen carbonate or the like is added to the above-mentioned reaction mixture to perform neutralization, thereby obtaining a sodium salt thereof, or an aqueous solution of potassium hydroxide, potassium carbonate, potassium hydrogen carbonate or the like is added to the above-mentioned reaction mixture to perform neutralization, thereby obtaining a potassium salt thereof, which salt is then subjected to chromatography. In the chromatography, SEPHADEX LH 20, anion exchange resins, polystyrene based adsorption resins (DIAION HP 20, AMBERLITE XAD 2 and the like), and reversed phase silica gels (ODS silica gel, octylsilica gel and the like) can be used.
In either case, the chromatographic fractionation can be carried out while confirming the separation by means of a high-performance liquid chromatography. The confirmation of separation can be carried out by monitoring an ultraviolet absorption with use of, for example, ODS silica gel column and a buffer solution or a mixture of an aqueous acid solution with methanol or acetonitrile as an eluent.
For example, it has been confirmed that with respect to a sodium salt of a compound of the general formula (VIII), wherein Z is benzyloxycarbonyl, when subjected to elution by means of chromatography using DIAION HP 20 as a packing according to a linear concentration gradient method wherein a constitution ratio of eluant goes from water to methanol, both isomers of the compound of the general formula (VIII) in each sodium salt can be separated from each other at a methanol concentration between 20 and 60%, though the separation is not complete.
Both stereo isomers of the compound of the general formula (VIII) are subjected to removal of the amino group-protecting group Z according to a conventional method, thereby obtaining a compound of the formula (V). Any means of removing the protecting group different depending upon the kind of the protecting group is known in the art. For example, a benzyloxycarbonyl group, a t-butoxycarbonyl group, a p-methoxybenzyloxycarbonyl group and a phthaloyl group can be removed by means of a catalytic reduction, an acid, a catalytic reduction or an acid, and a hydrazine decomposition, respectively. 
Among the thus obtained stereo isomers of the compound of the formula (V), a substance has been found to perfectly agree with the above-mentioned sulphostin obtained by the cultivation of microorganisms on the basis of behavior thereof in chromatography, physico-chemical properties thereof and enzyme inhibition activities mentioned below, which substance was obtained by subjecting the compound of the formula (VIII) having a benzyloxycarbonyl group to chromatography using DIAION HP 20, and further subjecting a later eluate to catalytic reduction to remove the benzyloxycarbonyl group. Thereby, a synthetic chemical process for producing sulphostin in accordance with the present invention has been attained.
In a manner similar to that in the synthesis of sulphostin mentioned above, two stereo isomers of the sulphostin have been obtained from D-ornithine.
With respect to a compound of the general formula (IV) wherein n is 0, namely a 4-membered compound, it can be obtained by using known 4-(carbobenzoxyamino-2-azetidinone) as it is.
Furthermore, compounds of general formulas (IX), (X), (XI) and (XII) have been obtained similarly from 2-aminocaprolactam and methyl L-2,4-diaminobutyrate obtained through Hofmann degradation of methyl N-protected glutamate. 
In the formula, n is 0, 1 or 3, and Z is the same amino group-protecting group as in the general formula (VI). 
In the formula, n is 0, 1 or 3, and Z is the same amino group-protecting group as in the general formula (VI). 
In the formula, n is 0, 1 or 3, Z is the same amino group-protecting group as in the general formula (VI), and M+ is the same monovalent cation as in the general formula (VIII). 
In the formula, n is 0, 1 or 3.
Since the compound of the general formula (XI) has been found to be a mixture of diastereoisomers like in the case of sulphostin, respective isomers have been chromatographically separated. The compound of the general formula (XI) separated has been subjected to removal of the protecting group, thereby obtaining a stereo isomer of the compound of the general formula (XII).